Drugs and reagents
Preparation of PHI via phillyrin hydrolysis: Naturally occurring
phillygenin (from an in-house natural product library, >
97% purity), Synthetic phillygenin (> 98% purity) forin vivo study from phillyrin hydrolysis (FigS1 to S5). The
extraction of PHI was extracted, identified, and characterized. Briefly,
Phillyrin (3.0 g, 5.62 mmol) was dissolved in 120 ml of methanol in a
250 ml of flask, to this solution was added 2 M HCl (24 ml, 12 mmol, 8.6
eq.). The reaction was refluxed at 65oC overnight. The
reaction mixture was cooled to room temperature and adjust pH to
5~6 with sat. NaHCO3, the resulting
mixture was evaporated under reduced pressure and then was extracted
with EtOAc (100 ml) three times. The organic phase was washed with brine
(100 ml) two times, dried with anhydrous MgSO4, and then evaporated. The
residue was subjected to a silica gel column eluting with
dichloromethane/methanol (20/1) to yield PHI (1.58 g, 4.25 mmol, 76%).
White solid, ESI-MS m/z (m): 373.3 [M +
H]+, 371.2 [M – H]-;1H NMR (500 MHz, CDCl3): δ6.94-6.84 (6H, m, Ar-H), 5.59 (1H, s, 4’-OH), 4.87 (1H, d, J =
6.6 Hz, H-7), 4.43 (1H, d, J = 7.2 Hz, H-7’), 4.13 (1H, dd,J = 1.3, 9.5 Hz, H-9’ax), 3.91 (3H, s, 3-OMe), 3.90 (3H, s,
3’-OMe), 3.89 (3H, s, 4-OMe), 3.86 (2H, m, H-9eq and H-9’eq), 3.33 (2H,
m, H-8, H-9ax), 2.91 (1H, m, H-8’); 13C NMR (125 MHz,
CDCl3) δ 148.8 (C-3), 148.0 (C-4), 146.7 (C-3’),
145.3 (C-4’), 133.0 (C-1’), 131.0 (C-1), 119.2 (C-6’), 117.7 (C-6),
114.2 (C-5’), 111.0 (C-5), 109.0 (C-2), 108.5 (C-2’), 87.7 (C-7’), 82.0
(C-7), 71.0 (C-9’), 69.7 (C-9), 55.9 (3 × OMe), 54.5 (C-8’), 50.2 (C-8).
Dextran Sulfate Sodium (DSS, MP Biomedicals); Urine fecal occult blood
test kit (Nanjing Jiancheng Bioengineering Institute); Goat anti-rabbit
Alexa Fluor 488 conjugate (Abcam); DAPI (Sigma-Aldrich); RNA simple
total RNA kit (Tiangen Biotech); Hifair Ⅱ 1st Strand cDNA Synthesis
SuperMix for qPCR (gDNA digester plus, Yeasen), Hieff qPCR SYBR Green
Master Mix (High Rox, Yeasen); Protease inhibitor cocktail (Roche Life
Science); BCA protein assay kit (Thermo Fisher Scientific);
2,4,6-trinitro-Benzenesulfonic acid (TNBS, Sigma-Aldrich); Antibodies
against Occludin, TLR4 (Abcam); Antibodies against E-cadherin, NF-κB,
p-NF-κB, Cleaved Caspase-1, MyD88, NLRP3, ASC (Cell Signaling
Technology); HRP-conjugated monoclonal mouse anti-GAPDH (KangChen);
Anti-mouse CD16/32 mAb, BV510-conjugated anti-CD45 mAb,
BUV395-conjugated anti-CD11b mAb, APC-conjugated anti-CD11c mAb,
PE-conjugated anti-Ly6G mAb, PerCP-Cy5.5-conjugated anti-CD3 mAb (BD
Bioscience); FITC-conjugated anti-F4/80 mAb (Thermo Fisher Scientific);
FITC-dextran 4KD (Sigma-Aldrich); FBS (Gibco); Colony-stimulating factor
(M-CSF, Peprotech); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazoliumbromide (MTT, Sigma-Aldrich); Lipopolysaccharide (LPS,
Sigma-Aldrich); Tissue protein extraction reagent (Thermo Fisher
Scientific); Mouse TNF-α, IL-6 (BD Biosciences); IL-1β ELISA kit (Thermo
Fisher Scientific); Phorbol myristate acetate (PMA, Sigama-Aldrich);
Adenosine triphosphate (ATP, Sigma-Aldrich).